Methods of Treating Cancer Using Compositions of Antibodies and Carrier Proteins with Antibody Pretreatment

ABSTRACT

Described herein are methods, formulations and kits for treating a patient with cancer with anti-VEGF antibodies and albumin-bound chemotherapeutic/anti-VEGF antibody nanoparticle complexes.

This application claims benefit of the filing dates of provisionalapplication Nos. 62/247,470 filed Oct. 28, 2015 and 62/252,377 filedNov. 6, 2015, the disclosures of which are incorporated herein byreference.

FIELD OF THE INVENTION

This disclosure relates to novel methods and kits for treating cancerusing vascular endothelial growth factor (VEGF) antibodies and carrierprotein/VEGF antibody complexes.

STATE OF THE ART

Chemotherapy remains a mainstay for systemic therapy for many types ofcancer, including melanoma. Most chemotherapeutics are only slightlyselective to tumor cells, and toxicity to healthy proliferating cellscan be high (Allen T M. (2002) Cancer 2:750-763), often requiring dosereduction and even discontinuation of treatment. In theory, one way toovercome chemotherapy toxicity issues as well as improve drug efficacyis to target the chemotherapy drug to the tumor using antibodies thatare specific for proteins selectively expressed (or overexpressed) bytumors cells to attract targeted drugs to the tumor, thereby alteringthe biodistribution of the chemotherapy and resulting in more drug goingto the tumor and less affecting healthy tissue. Despite 30 years ofresearch, however, specific targeting rarely succeeds in the therapeuticcontext.

Conventional antibody dependent chemotherapy (ADC) is designed with atoxic agent linked to a targeting antibody via a syntheticprotease-cleavable linker. The efficacy of such ADC therapy is dependenton the ability of the target cell to bind to the antibody, the linker tobe cleaved, and the uptake of the toxic agent into the target cell.Schrama, D. et al. (2006) Nature reviews. Drug discovery 5:147-159.

Antibody-targeted chemotherapy promised advantages over conventionaltherapy because it provides combinations of targeting ability, multiplecytotoxic agents, and improved therapeutic capacity with potentiallyless toxicity. Despite extensive research, clinically effectiveantibody-targeted chemotherapy remains elusive: major hurdles includethe instability of the linkers between the antibody and chemotherapydrug, reduced tumor toxicity of the chemotherapeutic agent when bound tothe antibody and the inability of the conjugate to bind and enter tumorcells. In addition, these therapies did not allow for control over thesize of the antibody-drug conjugates.

It was recently discovered that nanoparticle complexes of a carrierprotein-bound chemotherapeutic and antibody have superior therapeuticefficacy, including in humans, than either the chemotherapeutic orantibody delivered alone or sequentially, resulting in a significantlyreduced tumor size. See, PCT Application No. PCT/US 15/54295. It iscontemplated that these nanoparticles have improved targeting of thechemotherapeutics to the tumor, and that this targeting may be mediated,at least in part, by the bound antibody. It is further contemplated thatthe carrier protein-bound chemotherapeutic increases the targeting ofthe antibody to the tumor or that the existence of the complex showsgreater stability in vivo than chemotherapeutic alone.

Even still, there remains a need in the art to improve the efficacy ofcancer therapeutics.

SUMMARY

This invention is directed, in part, on the discovery that treatment ofa cancer expressing vascular endothelial growth factor (VEGF) with ananti-VEGF antibody composition enhances the effectiveness ofcarrier-bound (e.g., albumin) chemotherapeutic/anti-VEGF antibodynanoparticles containing a therapeutically effective amount of thechemotherapeutic. Preferably, such anti-VEGF antibodies are administeredprior to treatment with such nanoparticles. Accordingly, in one aspect,provided herein are methods for treating a patient suffering from acancer which expresses VEGF wherein said patient is treated with asub-therapeutic amount of an anti-VEGF antibody and carrier-bound (e.g.,albumin) chemotherapeutic/anti-VEGF antibody nanoparticle complexescontaining a therapeutically effective amount of the chemotherapeuticsuch that the administration of said sub-therapeutic amount of theanti-VEGF antibody enhances the efficacy of said nanoparticle complexes.It is contemplated that administration of a sub-therapeutic amount ofthe anti-VEGF antibody enhances the efficacy of the nanoparticlecomplexes, at least in part, by clearing the blood stream of at leastsome undesired, non-tumor VEGF targets. Treatment with a sub-therapeuticamount of anti-VEGF antibody may allow for greater targeting of thenanoparticle complexes to the tumor, decrease the amounts of thechemotherapeutic/antibody complexes administered to a patient, or both.

In another aspect, provided herein are methods for enhancing theefficacy of albumin-bound chemotherapeutic/anti-VEGF antibodynanoparticle complexes by administering the albumin-boundchemotherapeutic/anti-VEGF antibody nanoparticle complexes about 0.5 to48 hours after pretreatment of a patient with a sub-therapeutic amountof anti-VEGF antibody.

Preferably, such nanoparticle complexes are administered about 24 hoursafter the sub-therapeutic amount of anti-VEGF antibody.

In another aspect, provided herein are methods for enhancing thetherapeutic outcome in a patient suffering from a cancer expressingsoluble VEGF which patient is selected to be treated with nanoparticlescomprising albumin-bound paclitaxel and anti-VEGF antibodies whereinsaid antibodies of the nanoparticles are integrated onto and/or intosaid nanoparticles which method comprises treating said patient with asub-therapeutic amount of said anti-VEGF antibody prior to anysubsequent treatment with the nanoparticles.

In another aspect, provided herein are methods for enhancing thetherapeutic outcome in a patient suffering from a cancer overexpressingsoluble VEGF, said method comprising treating the patient with asub-therapeutic amount of said anti-VEGF antibody and co-treating saidpatients with an effective amount of nanoparticles comprisingalbumin-bound paclitaxel and anti-VEGF antibodies wherein saidantibodies of the nanoparticles are integrated onto and/or into saidnanoparticles.

In one embodiment, the chemotherapeutic is paclitaxel.

In one embodiment, the anti-VEGF antibody is bevacizumab or a biosimilarversion thereof.

In one embodiment, the sub-therapeutic amount of anti-VEGF antibody isselected from an amount consisting of about 1%, about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55% or about 60% of the therapeutic dosage ofanti-VEGF antibody. It is contemplated that administration of thesub-therapeutic amount of anti-VEGF antibody preferentially blockscirculating VEGF with minimal blocking of VEGF associated with a tumor).In some embodiments, the sub-therapeutic amount of anti-VEGF to beadministered to the patient is determined by analyzing the level ofcirculating VEGF in the blood.

In one embodiment, the sub-therapeutic amount of anti-VEGF antibody isadministered from between about 30 minutes to about 48 hours prior toadministration of the albumin-bound chemotherapeutic/anti-VEGF antibodynanoparticle complexes.

In one embodiment, the target of said nanoparticle complexes is a solidcancer.

In other aspects, provided herein are unit-dose formulations of ananti-VEGF antibody, for example, bevacizumab or a biosimilar versionthereof, which formulation comprises from about 1% to about 60% of atherapeutic dose of said antibody wherein said formulation is packagedso as to be administered as a unit dose.

In some embodiments, the formulation comprises from about 5% to about20% of a therapeutic dose of bevacizumab or a biosimilar versionthereof. The therapeutic dose for bevacizumab for a given approvedindication, e.g., treatment for metastatic colorectal cancer,non-squamous non-small cell lung cancer, metastatic breast cancer,glioblastoma, and metastatic renal cell carcinoma, is recited in theprescribing information. In each case the therapeutic dose is from 5 to15 mg/kg and preferably a subtherapeutic dose ranges from 5% to 20% ofthe therapeutic dose. In such a preferred embodiment such asubtherapeutic dose would range from 0.25 mg/kg to 3 mg/kg, morepreferably from 0.5 to 2 mg/kg.

In other aspects, provided herein are kits comprising: (a) an amount ofan albumin-bound chemotherapeutic/anti-VEGF antibody complexes, (b) aunit dose of a sub-therapeutic amount of anti-VEGF antibody, andoptionally (c) instructions for use.

In one embodiment, the albumin-bound chemotherapeutic/anti-VEGF antibodycomplexes of the kits are lyophilized.

An embodiment of the invention includes a method for increasing theduration of tumor uptake of a chemotherapeutic agent by administeringthe chemotherapeutic agent in a nanoparticle comprising a carrierprotein and the chemotherapeutic agent and having surface complexationwith an antibody, e.g., an antibody that specifically binds to anantigen on or shed by the tumor. In some embodiments the subjectreceives a subtherapeutic amount of the antibody prior to orconcurrently with such nanoparticles.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are representative only of the invention and arenot intended as a limitation. For the sake of consistency, thenanoparticles of this invention using albumin-bound paclitaxel (e.g.,ABRAXANE®) and bevacizumab employ the acronym “AB” and the number afterAB such as AB160 is meant to confer the average particle size of thesenanoparticles (in nanometers).

FIGS. 1A-E show in vivo testing of athymic nude mice injected with 1×106A375 human melanoma cells in the right flank and treated with (FIG. 1A)PBS, (FIG. 1B) 12 mg/kg BEV, (FIG. 1C) 30 mg/kg ABX, (FIG. 1D) AB160, or(FIG. 1E) pretreated with 1.2 mg/kg BEV and, 24 hr later, AB160. Data isrepresented at day 15-post treatment, or longer, as tumor volume in mm³.

FIG. 1F summarizes the day 7-post treatment data from FIGS. 1A-E.

FIG. 1G summarizes the day 10-post treatment data from FIGS. 1A-E.

FIG. 2 depicts the tumor measurements on day 15-post treatment witheither saline (PBS), bevacizumab (BEV), ABRAXANE® (ABX), AB160 orco-treatment with BEV one day (24 hours) prior to administration ofAB160 (BEV+AB160).

FIG. 3 depicts the tumor measurements on day 20-post treatment witheither saline (PBS), bevacizumab (BEV), ABRAXANE® (ABX), AB160 orco-treatment with BEV one day (24 hours) prior to administration ofAB160 (BEV+AB160).

FIG. 4 depicts the median survival with either saline (PBS), bevacizumab(BEV), ABRAXANE® (ABX), AB160 or co-treatment with BEV one day (24hours) prior to administration of AB160 (BEV+AB160).

FIG. 5 is fluorescent imagery of mice treated with AB160 with or withouta bevacizumab (BEV) pre-treatment. The fluorescent imagery was performedat an excitation/emission spectrum of 710/760.

FIG. 6, depicts the median survival with either saline (PBS),bevacizumab (BEV), ABRAXANE® (ABX), AB160 alone, AB225 and co-treatmentwith BEV prior to administration of AB160 (BEV+AB160).

FIG. 7 depicts fluorescence over time of AlexaFluor 750 labelednanoparticles in the tumors of mice treated with ABRAXANE® or AB160 withor without a pretreatment with bevacizumab. FIG. 7A depicts fluorescenceper unit area of tumor ROI's (upper three lines) and background ROIs(lower three lines) for all three treatment groups at 24, 29 and 48hours post injection. The dotted line represents an extrapolation offluorescence, indicating an expected complete clearance at about 72hours. FIG. 7B depicts the clearance from tumor ROIs adjusted forbackground fluorescence. FIG. 7C is a table of the clearance rates offluorescent nanoparticles indicating AB160 nanoparticles are clearednearly twice as slowly as ABRAXANE®.

DETAILED DESCRIPTION

After reading this description it will become apparent to one skilled inthe art how to implement the invention in various alternativeembodiments and alternative applications.

However, all the various embodiments of the present invention will notbe described herein. It will be understood that the embodimentspresented here are presented by way of an example only, and notlimitation. As such, this detailed description of various alternativeembodiments should not be construed to limit the scope or breadth of thepresent invention as set forth below.

Before the present invention is disclosed and described, it is to beunderstood that the aspects described below are not limited to specificcompositions, methods of preparing such compositions, or uses thereof assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

The detailed description of the invention is divided into varioussections only for the reader's convenience and disclosure found in anysection may be combined with that in another section. Titles orsubtitles may be used in the specification for the convenience of areader, which are not intended to influence the scope of the presentinvention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In this specification and inthe claims that follow, reference will be made to a number of terms thatshall be defined to have the following meanings:

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, concentration, and such other, including arange, indicates approximations which may vary by (+) or (−) 10%, 5%,1%, or any subrange or subvalue there between. Preferably, the term“about” when used with regard to a dose amount means that the dose mayvary by +/−10%.

“Comprising” or “comprises” is intended to mean that the compositionsand methods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination for the stated purpose. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed invention.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

As used herein, the term “sub-therapeutic” is used to describe an amountof antibody that is below the amount of antibody conventionally used totreat a cancer. For example, a sub-therapeutic amount is an amount lessthan that defined by the manufacturer as being required for therapy.

The term “nanoparticle” as used herein refers to particles having atleast one dimension which is less than 5 microns. In preferredembodiments, such as for intravenous administration, the nanoparticle isless than 1 micron. For direct administration, e.g., into a tumor, thenanoparticle can be larger. Even larger particles are expresslycontemplated by the invention.

In a population of particles, the size of individual particles aredistributed about a mean. Particle sizes for the population cantherefore be represented by an average, and also by percentiles. D50 isthe particle size below which 50% of the particles fall. 10% ofparticles are smaller than the D10 value and 90% of particles aresmaller than D90. Where unclear, the “average” size is equivalent toD50. So, for example, AB160 refers to nanoparticles having an averagesize of 160 nanometers.

The term “nanoparticle” may also encompass discrete multimers of smallerunit nanoparticles. For example, a 320 nm particle comprises a dimer ofa unit 160 nm nanoparticle. For 160 nm nanoparticles, multimers wouldtherefore be approximately 320 nm, 480 nm, 640 nm, 800 nm, 960 nm, 1120nm, and so on as determined by a Mastersizer 2000 (available fromMalvern Instruments Ltd, Wocestershire, UK) as described inPCT/US15/54295.

The term “biosimilar” as used herein refers to a biopharmaceutical whichis deemed to be comparable in quality, safety, and efficacy to areference product marketed by an innovator company (Section 351(i) ofthe Public Health Service Act (42 U.S.C. 262(i)).

The term “carrier protein” as used herein refers to proteins thatfunction to transport antibodies and/or therapeutic agents. Theantibodies of the present disclosure can reversibly bind to the carrierproteins. Exemplary carrier proteins are discussed in more detail below.

The term “core” as used herein refers to central or inner portion of thenanoparticle which may be comprised of a carrier protein, a carrierprotein and a therapeutic agent, or other agents or combination ofagents. In some embodiments, a hydrophobic portion of the antibody maybe incorporated into the core.

As used herein, the term “enhancing the therapeutic outcome” and thelike relative to a cancer patient refers to a slowing or diminution ofthe growth of cancer cells or a solid tumor, or a reduction in the totalnumber of cancer cells or total tumor burden.

The term “therapeutic agent” as used herein means an agent which istherapeutically useful, e.g., an agent for the treatment, remission orattenuation of a disease state, physiological condition, symptoms, oretiological factors, or for the evaluation or diagnosis thereof. Atherapeutic agent may be a chemotherapeutic agent, for example, mitoticinhibitors, topoisomerase inhibitors, steroids, anti-tumor antibiotics,antimetabolites, alkylating agents, enzymes, proteasome inhibitors, orany combination thereof.

The term “antibody” or “antibodies” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules (i.e., molecules that contain an antigenbinding site that immuno-specifically bind an antigen). The term alsorefers to antibodies comprised of two immunoglobulin heavy chains andtwo immunoglobulin light chains as well as a variety of forms includingfull length antibodies and portions thereof including, for example, animmunoglobulin molecule, a monoclonal antibody, a chimeric antibody, aCDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, aFv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), adiabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, a functionally activeepitope-binding fragment thereof bifunctional hybrid antibodies (e.g.,Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains(e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883(1988) and Bird et al., Science 242, 423-426 (1988), which areincorporated herein by reference). (See, generally, Hood et al.,Immunology, Benjamin, N.Y., 2ND ed. (1984); Harlow and Lane, Antibodies.A Laboratory Manual, Cold Spring Harbor Laboratory (1988); Hunkapillerand Hood, Nature, 323, 15-16 (1986), which are incorporated herein byreference). The antibody may be of any type (e.g., IgG, IgA, IgM, IgE orIgD). Preferably, the antibody is IgG. An antibody may be non-human(e.g., from mouse, goat, or any other animal), fully human, humanized,or chimeric. In an embodiment the antibody is an exogenous antibody. Anexogenous antibody is an antibody not naturally produced in a mammal,e.g. in a human, by the mammalian immune system.

The term “dissociation constant,” also referred to as “Kd,” refers to aquantity expressing the extent to which a particular substance separatesinto individual components (e.g., the protein carrier, antibody, andoptional therapeutic agent).

The terms “lyophilized,” “lyophilization” and the like as used hereinrefer to a process by which the material (e.g., nanoparticles) to bedried is first frozen and then the ice or frozen solvent is removed bysublimation in a vacuum environment. An excipient is optionally includedin pre-lyophilized formulations to enhance stability of the lyophilizedproduct upon storage. In some embodiments, the nanoparticles can beformed from lyophilized components (carrier protein, antibody andoptional therapeutic) prior to use as a therapeutic. In otherembodiments, the carrier protein, antibody, and optional therapeuticagent are first combined into nanoparticles and then lyophilized. Thelyophilized sample may further contain additional excipients.

The term “buffer” encompasses those agents which maintain the solutionpH in an acceptable range prior to lyophilization and may includesuccinate (sodium or potassium), histidine, phosphate (sodium orpotassium), Tris(tris(hydroxymethyl)aminomethane), diethanolamine,citrate (sodium) and the like. The buffer of this invention has a pH inthe range from about 5.5 to about 6.5; and preferably has a pH of about6.0. Examples of buffers that will control the pH in this range includesuccinate (such as sodium succinate), gluconate, histidine, citrate andother organic acid buffers.

The term “pharmaceutical formulation” refers to preparations which arein such form as to permit the active ingredients to be effective, andwhich contains no additional components which are toxic to the subjectsto which the formulation would be administered.

“Pharmaceutically acceptable” excipients (vehicles, additives) are thosewhich can reasonably be administered to a subject mammal to provide aneffective dose of the active ingredient employed.

“Reconstitution time” is the time that is required to rehydrate alyophilized formulation into a solution.

A “stable” formulation is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage.

The term “epitope” as used herein refers to the portion of an antigenwhich is recognized by an antibody. Epitopes include, but are notlimited to, a short amino acid sequence or peptide (optionallyglycosylated or otherwise modified) enabling a specific interaction witha protein (e.g., an antibody) or ligand. For example, an epitope may bea part of a molecule to which the antigen-binding site of an antibodyattaches.

The term “treating” or “treatment” covers the treatment of a disease ordisorder (e.g., cancer), in a subject, such as a human, and includes:(i) inhibiting a disease or disorder, i.e., arresting its development;(ii) relieving a disease or disorder, i.e., causing regression of thedisorder; (iii) slowing progression of the disorder; and/or (iv)inhibiting, relieving, or slowing progression of one or more symptoms ofthe disease or disorder. In some embodiments “treating” or “treatment”refers to the killing of cancer cells.

The term “kill” with respect to a cancer treatment is directed toinclude any type of manipulation that will lead to the death of thatcancer cell or at least a portion of a population of cancer cells.

The term “dose” refers to an amount of antibody given to a patient inneed thereof. The attending clinician will select an appropriate dosefrom the range based on the patient's weight, age, health, stage ofcancer, level of circulating VEGF, and other relevant factors, all ofwhich are well within the skill of the art.

The term “unit dose” refers to a dose of the antibody that is given tothe patient to provide a desired result. In some instances, the unitdose is sold in a sub-therapeutic formulation (e.g., 10% the therapeuticdose). The unit dose may be administered as a single dose or a series ofsubdoses. The therapeutic dose for an antibody for a given FDA-approvedindication is recited in the prescribing information, for example thetherapeutic dose of bevacizumab is 5 mg/kg to 15 mg/kg depending on thecondition, and preferably a subtherapeutic dose ranges from 5% to 20% ofthe therapeutic dose. In such a preferred embodiment such asubtherapeutic dose would range from 0.25 mg/kg to 3 mg/kg, morepreferably from 0.5 to 2 mg/kg. The therapeutic dose for an antibody fora given indication where the antibody is not yet FDA approved or theantibody is not yet approved for that indication, will be the amount thecorrelates to the therapeutic that has been approved for otherindications, and thus the subtherapeutic dose for the non-FDA approvedindications is readily calculated as a percent of the therapeutic dose(e.g., 10% of the therapeutic dose). For example, the therapeutic doseand therefore the subtherapeutic dose of an antibody for the treatmentof metastatic melanoma correlates to the therapeutic dose for metastaticcancers in general that has been approved.

Additionally, some terms used in this specification are morespecifically defined below.

Overview

The current invention is predicated, in part, on the surprisingdiscovery that treatment of a cancer in a patient with an anti-VEGFantibody composition together with albumin-boundchemotherapeutic/anti-VEGF antibody nanoparticle complexes provides forunexpectedly improved therapeutic outcomes.

As will be apparent to the skilled artisan upon reading this disclosure,the present disclosure relates to methods for treating a patientsuffering from a cancer by treating the patient with a sub-therapeuticamount of an anti-VEGF antibody and albumin-boundchemotherapeutic/anti-VEGF antibody nanoparticle complexes containing atherapeutically effective amount of the chemotherapeutic.

Anti-VEGF Antibodies

In some embodiments, the anti-VEGF antibody is bevacizumab or abiosimilar version thereof

Bevacizumab (AVASTTN®, Roche, USA) is a humanized monoclonal antibodythat inhibits angiogenesis by blocking the action of vascularendothelial growth factor (VEGF). As such, bevacizumab can slow thegrowth of new blood vessels in tumors thereby inhibiting the tumorsability to grow. Bevacizumab has been used to treat various cancersincluding, non-small cell lung cancer (NSCLC), metastatic colorectalcancer (mCRC), platinum-resistant ovarian cancer (prOC), advancedcervical cancer (CC), metastatic renal cell carcinoma (mRCC), andrecurrent glioblastoma (rGBM). Several biosimilar versions ofbevacizumab are currently being developed including ABP 215(Amgen/Allergen, USA), BCD-021 (Biocad, Russia), BI 695502 (BoehringerIngelheim, Germany), and PF-06439535 (Pfizer, USA), among others.

In some embodiments, the sub-therapeutic amount of anti-VEGF antibody isselected from an amount consisting of about 1%, about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55% or about 60% of the therapeutic dosage ofanti-VEGF antibody.

In some embodiments, the sub-therapeutic amount of anti-VEGF antibody isan amount which preferentially blocks circulating VEGF without blockingVEGF associated with tumor.

Complexes

Carrier protein-bound chemotherapeutic/anti-VEGF antibody complexes andmethods of preparing the complexes are described, for example, in U.S.Provisional App. No. 62/060,484, filed Oct. 6, 2014; and U.S.Provisional Patent Application Nos. 62/206,770; 62/206,771; and62/206,772 filed Aug. 18, 2015, as well as PCT Publication No.PCT/US15/54295, filed Oct. 6, 2015. The contents of each of theseapplications are specifically incorporated by reference in theirentireties. Example 1 below provides one example of a detailed protocolfor making such complexes.

In some embodiments, the anti-VEGF antibody is bevacizumab or abiosimilar version thereof. In some embodiments, the antibodies are asubstantially single layer of antibodies on all or part of the surfaceof the nanoparticle.

In some embodiments, the at least one chemotherapeutic agent is selectedfrom the group consisting of abiraterone, bendamustine, bortezomib,carboplatin, cabazitaxel, cisplatin, chlorambucil, dasatinib, docetaxel,doxorubicin, epirubicin, erlotinib, etoposide, everolimus, gefitinib,idarubicin, imatinib, hydroxyurea, imatinib, lapatinib, leuprorelin,melphalan, methotrexate, mitoxantrone, nedaplatin, nilotinib,oxaliplatin, paclitaxel, pazopanib, pemetrexed, picoplatin, romidepsin,satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin,vinblastine, vinorelbine, vincristine, and cyclophosphamide. Inpreferred embodiments, the chemotherapeutic is paclitaxel.

In some embodiments, the nanoparticle sizes are between 200 and 800 nm,including 200, 300, 400, 500, 600, 700 or 800 nm. In other embodiments,the nanoparticles are larger, e.g. from greater than 800 nm to about 3.5μm. In some embodiments, the particles are multimers of nanoparticles.In some embodiments the nanoparticles have average particle sizes ofabout 160 nm to about 225 nm either freshly made or after lyophilizationand resuspension in an aqueous solution suitable for injection.

Treatment Methods

In one aspect is provided a method for treating a patient suffering froma cancer which expresses VEGF wherein said patient is treated with asub-therapeutic amount of an anti-VEGF antibody and albumin-boundchemotherapeutic/anti-VEGF antibody nanoparticle complexes containing atherapeutically effective amount of the chemotherapeutic such that theadministration of said sub-therapeutic amount of the anti-VEGF antibodyenhances the efficacy of said nanoparticle complexes.

For the sake of clarification, “co-treatment” refers to treatment of thecancer expressing VEGF (a soluble cytokine) with an anti-VEGF antibodyprior, concurrent or immediately after administration of thealbumin-bound chemotherapeutic/anti-VEGF antibody complex provided thatthe anti-VEGF antibody is capable of preferentially binding solubleVEGF.

In one embodiment, the anti-VEGF antibody is administered in asub-therapeutic dose prior to the nanoparticle complex. In thisembodiment, the administration of the anti-VEGF antibody occurs about0.5 to 48 hours prior to administration of the nanoparticle complexes.

In another embodiment, the anti-VEGF antibody composition isadministered between 0.5 hours prior to and up to 0.5 hours afteradministration of the nanoparticle complexes. In this embodiment, it iscontemplated that such administration will nevertheless result inbinding of some of the circulating VEGF by the antibody composition.

In yet another embodiment, the antibody composition can be administeredup to 2 hours post administration of the nanoparticle complexes.

In a preferred aspect, there is provided methods for enhancing theefficacy of albumin-bound chemotherapeutic/anti-VEGF antibodynanoparticle complexes by administering the albumin-boundchemotherapeutic/anti-VEGF antibody nanoparticle complexes about 0.5 to48 hours after pretreatment of a patient with a sub-therapeutic amountof anti-VEGF antibody. Preferably, such nanoparticle complexes areadministered about 24 hours after the sub-therapeutic amount ofanti-VEGF antibody.

In another aspect, there is provided methods for enhancing thetherapeutic outcome in a patient suffering from a cancer expressingsoluble VEGF which patient is selected to be treated with nanoparticlescomprising albumin-bound paclitaxel and anti-VEGF antibodies whereinsaid antibodies of the nanoparticles are integrated onto and/or intosaid nanoparticles which method comprises treating said patient with asub-therapeutic amount of said anti-VEGF antibody prior to anysubsequent treatment with the nanoparticles.

In another aspect, there is provided methods for enhancing thetherapeutic outcome in a patient suffering from a cancer overexpressingsoluble VEGF, said method comprising treating the patient with asub-therapeutic amount of said anti-VEGF antibody and co-treating saidpatients with an effective amount of nanoparticles comprisingalbumin-bound paclitaxel and anti-VEGF antibodies wherein saidantibodies of the nanoparticles are integrated onto and/or into saidnanoparticles.

In another aspect, there is provided a method for enhancing thetherapeutic outcome in a patient suffering from a cancer expressingsoluble VEGF which patient is to be treated with nanoparticlescomprising albumin-bound paclitaxel and anti-VEGF antibodies whereinsaid antibodies of the nanoparticles are integrated onto and/or intosaid nanoparticles which method comprises treating said patient with asub-therapeutic amount of said anti-VEGF antibody within +/−0.5 hours ofadministration of said nanoparticles.

In another aspect is provided a method for enhancing the therapeuticoutcome in a patient suffering from a cancer overexpressing soluble VEGFwhich patient has been treated with a sub-therapeutic amount of saidanti-VEGF antibody said method comprising treating said patients with aneffective amount of nanoparticles comprising albumin-bound paclitaxeland anti-VEGF antibodies wherein said antibodies of the nanoparticlesare integrated onto and/or into said nanoparticle antibody within +/−0.5hours of administration of said antibodies.

The patient may be co-treated with a sub-therapeutic amount of ananti-VEGF antibody and albumin-bound chemotherapeutic/anti-VEGF antibodycomplex.

In some embodiments the anti-VEGF antibody is administered prior to thealbumin-bound chemotherapeutic/anti-VEGF antibody complex, for example,the anti-VEGF antibody can be administered minutes, hours or days priorto administration of the albumin-bound chemotherapeutic/anti-VEGFantibody complex. In some embodiments, the anti-VEGF antibody isadministered between about 5 to about 59 minutes, about 10 to about 50minutes, about 15 to about 45 minutes, about 20 to about 40 minutes,about 25 to about 35 minutes prior to administration of thealbumin-bound chemotherapeutic/anti-VEGF antibody complex. In otherembodiments, the anti-VEGF antibody can be administered about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours,or longer prior to administration of the albumin-boundchemotherapeutic/anti-VEGF antibody complex. In other embodiments, theanti-VEGF antibody can be administered about 1 day, about 2 days, about3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10days, about 12 days, about 15 days, or longer prior to administration ofthe albumin-bound chemotherapeutic/anti-VEGF antibody complex.

In some embodiments, the anti-VEGF antibody can be administeredconcurrently with administration of the albumin-boundchemotherapeutic/anti-VEGF antibody complex, for example, within 10minutes or less of each other.

In other embodiments, the anti-VEGF antibody can be administeredsubsequent to administration of the albumin-boundchemotherapeutic/anti-VEGF antibody complex, for example, within 2 hoursafter administration of the albumin-bound chemotherapeutic/anti-VEGFantibody complex, provided that the subsequent administration allows theantibody to preferentially bind the soluble VEGF.

Cancers or tumors that can be treated by the compositions and methodsdescribed herein include, but are not limited to: biliary tract cancer;brain cancer, including glioblastomas and medulloblastomas; breastcancer; cervical cancer; choriocarcinoma; colon cancer; endometrialcancer; esophageal cancer, gastric cancer; hematological neoplasms,including acute lymphocytic and myelogenous leukemia; multiple myeloma;AIDS associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms, including Bowen's disease and Paget'sdisease; liver cancer (hepatocarcinoma); lung cancer; lymphomas,including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas;oral cancer, including squamous cell carcinoma; ovarian cancer,including those arising from epithelial cells, stromal cells, germ cellsand mesenchymal cells; pancreas cancer; prostate cancer; rectal cancer;sarcomas, including leiomyo sarcoma, rhabdomyosarcoma, liposarcoma,fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi'ssarcoma, basocellular cancer and squamous cell cancer; testicularcancer, including germinal tumors (seminoma, non-seminoma[teratomas,choriocarcinomas]), stromal tumors and germ cell tumors; thyroid cancer,including thyroid adenocarcinoma and medullar carcinoma; and renalcancer including adenocarcinoma and Wilms tumor. In importantembodiments, cancers or tumors include breast cancer, lymphoma, multiplemyeloma, and melanoma.

Formulations

In one aspect, the anti-VEGF is a unit-dose formulation of an anti-VEGFantibody which formulation comprises from about 1% to about 60% of atherapeutic dose of said antibody wherein said formulation is packagedso as to be administered as a unit dose. In an aspect of the invention,the unit-dose formulation of an anti-VEGF antibody comprises about 10%of a therapeutic dose of said antibody. For example 10% of a therapeuticdose of an anti-VEGF antibody, e.g., bevacizumab, may be 0.5 mg/kg to 5mg/kg.

The unit-dose formulation of an anti-VEGF antibody can be about 1% toabout 60%, about 5% to about 50%, about 10% to about 40%, about 15% toabout 30%, about 20% to about 25%, of a therapeutic dose of theanti-VEGF antibody. Contemplated values include any value, subrange, orrange within any of the recited ranges, including endpoints.

In some embodiments, the anti-VEGF antibody is bevacizumab or abiosimilar version thereof, which formulation comprises from about 5% toabout 20% of a therapeutic dose of bevacizumab or a biosimilar versionthereof

In another aspect, provided herein is a formulation comprising ananti-VEGF antibody provided herein, and at least one pharmaceuticallyacceptable excipient.

In general, the unit-dose formulations provided herein can be formulatedfor administration to a patient by any of the accepted modes ofadministration. Various formulations and drug delivery systems areavailable in the art. See, e.g., Remington's Pharmaceutical Sciences,edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).

In general, unit-dose formulations provided herein will be administeredas pharmaceutical compositions by any one of the following routes: oral,systemic (e.g., transdermal, intranasal or by suppository), orparenteral (e.g., intramuscular, intravenous or subcutaneous)administration.

The unit-dose formulations may be comprised of in general, an anti-VEGFantibody, optionally in combination with at least one pharmaceuticallyacceptable excipient. Acceptable excipients are non-toxic, aidadministration, and do not adversely affect the therapeutic benefit ofthe claimed compounds. Such excipient may be any solid, liquid,semi-solid or, in the case of an aerosol composition, gaseous excipientthat is generally available to one of skill in the art.

Solid pharmaceutical excipients include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, animal, vegetable orsynthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil, etc. Preferred liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols. Othersuitable pharmaceutical excipients and their formulations are describedin Remington's Pharmaceutical Sciences, edited by E. W. Martin (MackPublishing Company, 18th ed., 1990).

The present formulations may, if desired, be presented in a pack ordispenser device containing a unit-dose of the active ingredient. Such apack or device may, for example, comprise metal or plastic foil, such asa blister pack, or glass, and rubber stoppers such as in vials. The packor dispenser device may be accompanied by instructions foradministration. Compositions comprising a unit-dose formulation of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

Kits

In some aspects, the current invention relates to kits comprising: (a)an amount of an albumin-bound chemotherapeutic/anti-VEGF antibodycomplexes, (b) a unit dose of a sub-therapeutic amount of anti-VEGFantibody, and optionally (c) instructions for use.

In some embodiments, the kits can include lyophilized complexes of thealbumin-bound chemotherapeutic/anti-VEGF antibody.

In some preferred embodiments, the kit components can be configure insuch a way that the components are accessed in their order of use. Forexample, in some aspects the kit can be configured such that uponopening or being accessed by a user, the first component available isthe unit dose of a sub-therapeutic amount of anti-VEGF antibody, forexample, in a first vial. A second container (e.g., a vial) comprisingor containing an amount of the albumin-bound chemotherapeutic/anti-VEGFantibody complexes can then be accessed. As such the kits can beintuitively configured in a way such that the first vial must be openedprior to the second vial being opened. It should be understood that insome embodiments, the order can be different, for example, where it isdesired to administer the complex first, prior to the administration ofthe antibody. Also, it can be configured such that both are administeredat the same time. Finally, it should be understood that additional vialsor containers of either or both component(s) can be included, andconfigured for opening in any desired order. For example, the first vialcould be antibody, the second vial could include complex, a third couldinclude either antibody or complex, etc. It is contemplated that a kitconfigured in such a way would prevent, or at least help to prevent, thecomponents from being administered in an order not intended by theinstructions for use.

In some aspects, the invention is directed to a kit of parts foradministration of albumin-bound chemotherapeutic/anti-VEGF antibodycomplexes and a unit dose of a sub-therapeutic amount of anti-VEGFantibody; and optionally further comprising a dosing treatment schedulein a readable medium. In some embodiments, the dosing schedule includesthe sub-therapeutic amount of anti-VEGF antibody required to achieve adesired average serum level is provided. In some embodiments, the kit ofparts includes a dosing schedule that provides an attending clinicianthe ability to select a dosing regimen of the sub-therapeutic amount ofanti-VEGF antibody based on the sex of the patient, mass of the patient,and the serum level that the clinician desires to achieve. In someembodiments, the dosing treatment is based on the level of circulatingVEGF in the blood of the patient. In some embodiments, the dosingschedule further provides information corresponding to the volume ofblood in a patient based upon weight (or mass) and sex of the patient.In an embodiment, the storage medium can include an accompanyingpamphlet or similar written information that accompanies the unit doseform in the kit. In an embodiment, the storage medium can includeelectronic, optical, or other data storage, such as a non-volatilememory, for example, to store a digitally-encoded machine-readablerepresentation of such information

The term “readable medium” as used herein refers to a representation ofdata that can be read, for example, by a human or by a machine.Non-limiting examples of human-readable formats include pamphlets,inserts, or other written forms. Non-limiting examples ofmachine-readable formats include any mechanism that provides (i.e.,stores and/or transmits) information in a form readable by a machine(e.g., a computer, tablet, and/or smartphone). For example, amachine-readable medium includes read-only memory (ROM); random accessmemory (RAM); magnetic disk storage media; optical storage media; andflash memory devices. In one embodiment, the machine-readable medium isa CD-ROM. In one embodiment, the machine-readable medium is a USB drive.In one embodiment, the machine-readable medium is a Quick Response Code(QR Code) or other matrix barcode.

EXAMPLES

The present disclosure is illustrated using a pre-treatment ofbevacizumab (i.e., AVASTIN®) followed by nanoparticles composed ofalbumin-bound paclitaxel (i.e., ABRAXANE®) and bevacizumab (i.e.,AVASTIN®).

One skilled in the art would understand that making and using thenanoparticles, as well as administration of a co-treatment ofbevacizumab, of the Examples are for the sole purpose of illustration,and that the present disclosure is not limited by this illustration.

Any abbreviation used herein, has normal scientific meaning. Alltemperatures are ° C. unless otherwise stated. Herein, the followingterms have the following meanings unless otherwise defined:

-   -   ABX=ABRAXANE®/(albumin-bound    -   ADC=antibody dependent chemotherapy    -   BEV=bevacizumab    -   BSA=bovine serum albumin    -   dH₂O=distilled water    -   kg=kilogram    -   nM=nano molar    -   mg=milligram    -   ml or mL=milliliter    -   m²=square meters    -   mm³=cubic millimeter    -   μg microgram    -   μl=microliter    -   μm=micrometer/micron    -   PBS=Phosphate buffered saline

Example 1 Nanoparticle Preparation

ABRAXANE® (ABX) (10 mg) was suspended in bevacizumab (BEV) (4 mg [160μl] unless otherwise indicated), and 840 μl of 0.9% saline was added togive a final concentration of 10 mg/ml and 2 mg/ml of ABX and BEV,respectively. The mixture was incubated for 30 minutes at roomtemperature (or at the temperature indicated) to allow particleformation. For Mastersizer experiments to measure particle size ofABX:BEV complexes, 10 mg of ABX was suspended in BEV at concentrationsof 0 to 25 mg/ml. Complexes of ABX with rituximab (0-10 mg/ml) ortrastuzumab (0-22 mg/ml) were formed under similar conditions.

For use in humans, the ABXBEV complexes may be prepared by obtaining thedose appropriate number of 4 mL vials of 25 mg/mL BEV and diluting eachvial per the following directions to 4 mg/mL. The dose appropriatenumber of 100 mg vials of ABX can be prepared by reconstituting to afinal concentration containing 10 mg/mL ABX nanoparticles. Using asterile 3 mL syringe, 1.6 mL (40 mg) of bevacizumab (25 mg/mL) can bewithdrawn and slowly injected, over a minimum of 1 minute, onto theinside wall of each of the vials containing 100 mg of ABX. Thebevacizumab solution should not be injected directly onto thelyophilized cake as this will result in foaming. Then, using a sterile12 mL sterile syringe, 8.4 mL 0.9% Sodium Chloride Injection, USP, canbe withdrawn and slowly injected, over a minimum of 1 minute, 8.4 mLonto the inside wall of each vial containing ABX 100 mg and BEV 40 mg.Once the addition of BEV 1.6 mL and 0.9% Sodium Chloride Injection, USP8.4 mL is completed, each vial can be gently swirled and/or invertedslowly for at least 2 minutes until complete dissolution of anycake/powder occurs. Generation of foam should be avoided. At this point,the concentration of each vial should be 100 mg/10 mL ABX and 40 mg/10mL BEV. The vials containing the ABX and BEV should sit for 60 minutes.The vial(s) should be gently swirled and/or inverted every 10 minutes tocontinue to mix the complex. After 60 minutes has elapsed, thecalculated dosing volume of ABX and BEV should be withdrawn from eachvial and slowly added to an empty viaflex bag. An equal volume of 0.9%Sodium Chloride Injection, USP is then added to make the finalconcentration of ABX 5 mg/mL and BEV 2 mg/mL. The bag should then begently swirled and/or inverted slowly for 1 minute to mix. The ABX:BEVnanoparticles can be stored for up to 4 hours at room temperaturefollowing final dilution.

Example 2 Co-Treatment with BEV Improves Targeting of ABX/BEV Complexes

Athymic nude mice were injected with 1×106 A375 human melanoma cells inthe right flank and then treated with PBS, 12 mg/kg BEV, 30 mg/kg ABXAB160, or pretreated with 1.2 mg/kg BEV and, 24 hr later, AB160. AB160was prepared as described in PCT Application No. PCT/US15/54295 andExample 1 above. FIGS. 1A-E track tumor size over 40 days (all mice inthe PBS, BEV, and ABX died by day 20). Data is represented as tumorvolume in mm³. Only mice treated with AB160 (with or withoutpretreatment with BEV) showed a reduction in average tumor volume. Seealso FIG. 1F and FIG. 1G.

The day 7-post treatment data, as summarized in FIG. 1F, show thatpretreatment with BEV was associated with a statistically significantreduction in tumor volume over control or BEV alone (p≦0.0001), or ABXalone (p≦0.0001).

The day 10-post treatment data, as summarized in FIG. 1G, again showthat pretreatment with BEV was associated with a statisticallysignificant reduction in tumor volume over control or BEV alone(p≦0.0001), or ABX alone (p≦0.0001). Pretreatment with BEV before AB160was also associated with a reduction in tumor volume over AB160 alone(p=0.02), with complete response in two mice.

In this experiment, a 12 mg/kg dose of BEV was not therapeutic. Theamount of BEV added in the pretreatment group was only 1.2 mg/kg, whichis 1/10 the usual dose in mice. Yet pretreatment with a subtherapeuticdose improved the efficacy of the AB160 nanoparticles. This data supportthe idea that pretreatment with a subtherapeutic amount of BEV can clearsystemic levels of VEGF, leaving a greater relative concentration at thetumor such that tumor-associated VEGF targeting by the AB160nanoparticles is more effective.

Tumors were measured on day 15 following treatment with either saline(PBS), AVASTIN® (BEV), ABRAXANE® (ABX), AB160 or a pretreatment of BEVone day before AB160 (BEV+AB160). A 10% sub-therapeutic dose of BEV, ascompared to the dose give to the BEV alone or AB160 cohort, was given tothe BEV+AB160 cohort 24 hours prior to administration of the AB160. TheBEV+AB160 cohort presented with delayed tumor growth, even when comparedto AB160, with two mice presenting with complete cures. FIG. 2. Assummarized in Table 1, these experiments also show that pre-treatmentwith BEV+AB160, increases survival. At day 15, 100% of the mice in theBEV+AB160 cohort were alive, whereas 86% of these mice in the AB160cohort were still alive. Both treatment groups had substantially greatersurvival than the PBS, BEV-only, or ABX-only treatment groups whichexhibited a 0%, 0% and 11% survival rate, respectively. At day 20, 92%of the mice in the BEV+AB160 cohort were alive, whereas 67% of the micein the AB160 cohort were still alive. FIG. 3.

TABLE 1 Percentage of mice alive on day 15 Treatment Group PBS BEV ABXAB160 BEV + AB160 Percent Alive on day 15 0% 0% 11% 86% 100%

Survival was again assessed at day 40. FIG. 4. As shown median survivalof both the mice treated with PBS or BEV was 7 days. Mice treated withABX alone shown median survival of 14 days, while those treated withAB160 exhibited a median survival of 25 days.

Median survival of mice co-treated with BEV and AB160 remainedundefined, with approximately 50% of the mice co-treated with BEV andAB160 still alive, at day 40.

Example 3 Fluorescence Over Time of AlexaFluor 750 Labeled Nanoparticles

Mice received IV injections of equal amounts of either labeledABRAXANE®, or nanoparticles of ABRAXANE® having surface complexationwith bevacizumab (BEV) as per Example 1 above (AB160); one AB160 groupof mice received a pre-treatment 1.2 mg/kg of bevacizumab. Fluorescentimagery was done at an excitation/emission spectrum of 710/760 (FIG. 5).Regions of interest (ROI) in the mice of FIG. 5 were assigned bysoftware to track tumor accumulation based on a fluorescence threshold.Fluorescence per unit area of background ROIs and tumor ROIs for allthree treatment groups were determined at 24, 29, and 48 hours postinjection

The amount of fluorescence (and thus paclitaxel) in the tumor andbackground ROIs at 24, 29 and 48 hour presented in FIGS. 7A and 7B. Thedata demonstrates that pretreatment with BEV results in higher levels oftumor fluorescence as compared AB160 alone or ABRAXANE alone.Furthermore, the clearance rate for AB160-treated mice with or without aBEV pre-treatment is nearly twice as slow as the ABRAXANE treated mice(FIG. 7C). These results evidence that pretreatment with BEV and use ofABRAXANE® nanoparticles having surface complexation with BEV providesfor a method for increasing the duration of tumor uptake of albumincontaining a chemotherapeutic agent both at 24 hours and 48 hours.Likewise, use of ABRAXANE® nanoparticles having surface complexationwith BEV also provides for increasing the duration of tumor uptake ofthese albumin containing nanoparticles with or without pretreatment withBEV at 48 hours. Both of these results evidence continued uptake for 48hours and possibly out to 72 hours. On the other hand, backgroundfluorescence of the corresponding nanoparticles is projected to go toabout 0 at 72 hours evidencing that systemic circulation of the drugshave disappeared from all parts of the animal but for the tumor. Alltold, the incorporation of the BEV into ABRAXANE® has a stabilizingimpact, which is most evident by the concentration of the drug in thetumor at 48 hours and projected for 72 hours. Without being limited toany theory, the antibody coating of the albumin nanoparticles impartsstability possibly by reducing liver or kidney clearance and/or byreducing protease degradation of the albumin carrier. This approachallows targeting antibodies to complex with a protein carrier such asalbumin and any chemotherapeutic drug, such as, e.g., abiraterone,bendamustine, bortezomib, carboplatin, cabazitaxel, cisplatin,chlorambucil, dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib,etoposide, everolimus, 5-fluoruracil, gefitinib, idarubicin, imatinib,hydroxyurea, imatinib, lapatinib, leuprorelin, melphalan, methotrexate,mitoxantrone, nedaplatin, nilotinib, oxaliplatin, paclitaxel, pazopanib,pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib,sunitinib, teniposide, triplatin, vinblastine, vinorelbine, vincristine,and cyclophosphamide thereby providing prolonged delivery of such drugsto the tumor.

FIG. 7A indicates that at 24 hours the total amount of fluorescenceaccumulating in the tumor of AB160-treated mice without a BEVpretreatment was less than that achieved in ABRAXANE®-treated mice.However, the rate of clearance of ABRAXANE® in treated mice was higherthan AB160 in treated mice. Therefore if one were to extrapolate thedata in FIG. 7A, as shown by the dotted line, the level of fluorescence(and thus paclitaxel) would be maintained at a higher level in thetumors of AB160-treated mice with or without BEV pre-treatment than thelevel maintained by ABRAXANE treatment.

FIG. 7 also demonstrates that the background fluorescence inAB160-treated mice with or without pretreatment is higher than that inABRAXANE-treated mice. While not wishing to be bound by theory, thissuggests that the antibodies confer additional stability to thenanoparticles of paclitaxel and albumin such that they are cleared moreslowly from the circulation than ABRAXANE alone. This would beadvantageous as it would provide higher levels of the nanoparticles forlonger periods of time for subsequent infiltration into the tumor.

Example 16 Nanoparticles Having a Size of 225 nm

To make a nanoparticle having a size of 225 nm, the particles wereprepared in accordance with Example 1 but the ratio of BEV to ABRAXANE®was 4:5, i.e., 4 parts BEV and 5 parts ABRAXANE. This ratio producednanoparticles having a size of 225 nm (AB225). The effect of AB225 wasassayed in animals as set forth above. FIG. 6 demonstrates that thesurvival of the mice treated with a single dose of saline, BEV24 (24mg/kg), ABX30(30 mg/kg), AB160 (12 mg/kg BEV and 30 mg/kg ABX) and AB225(24 mg/kg BEV and 30 mg/kg ABX) and with AB160 with a BEV (1.2 mg/kg)pretreatment. At 30 days the survival of mice treated with AB225, andwith AB160 with or without pretreatment with BEV far exceeded thesurvival of mice treated with BEV alone of ABRAXANE alone.

1. A method for treating a patient suffering from a cancer whichexpresses VEGF wherein said patient is treated with a sub-therapeuticamount of an anti-VEGF antibody and albumin-boundchemotherapeutic/anti-VEGF antibody nanoparticle complexes containing atherapeutically effective amount of the chemotherapeutic such that theadministration of said sub-therapeutic amount of the anti-VEGF antibodyenhances the efficacy of said nanoparticle complexes, wherein thesub-therapeutic amount of anti-VEGF antibody is administered frombetween about 30 minutes to about 48 hours prior to administration ofthe albumin-bound chemotherapeutic/anti-VEGF antibody nanoparticlecomplexes.
 2. The method of claim 1, wherein the chemotherapeutic ispaclitaxel.
 3. The method of claim 1, wherein the anti-VEGF antibody isbevacizumab or a biosimilar version thereof.
 4. The method of claim 1,wherein the sub-therapeutic amount of anti-VEGF antibody is selectedfrom an amount consisting of about 1%, about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55% or about 60% of the therapeutic dosage of anti-VEGFantibody.
 5. The method of claim 1, wherein the sub-therapeutic amountof anti-VEGF antibody is an amount which blocks circulating VEGF withoutblocking VEGF on a target of said nanoparticle complexes.
 6. The methodof claim 5, wherein the target of said nanoparticle complexes is a solidcancer.
 7. The method of claim 1, wherein the sub-therapeutic amount ofanti-VEGF to be administered to the patient is determined by analyzingthe level of circulating VEGF in the blood.
 8. (canceled)
 9. A methodfor enhancing the efficacy of albumin-bound chemotherapeutic/anti-VEGFantibody nanoparticle complexes by administering the albumin-boundchemotherapeutic/anti-VEGF antibody nanoparticle complexes about 24hours after administrating a sub-therapeutic amount of anti-VEGFantibody to a patient in need thereof.
 10. The method of claim 9,wherein the anti-VEGF antibody is bevacizumab or a biosimilar versionthereof.
 11. The method of claim 9, wherein the sub-therapeutic amountof anti-VEGF antibody is selected from an amount consisting of about 1%,about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55% or about 60% of thetherapeutic dosage of anti-VEGF antibody.
 12. The method of claim 9,herein the sub-therapeutic amount of anti-VEGF antibody is an amountwhich blocks circulating VEGF without blocking VEGF on a target of saidnanoparticle complexes.
 13. The method of claim 12, wherein the targetof said nanoparticle complexes is a solid cancer.
 14. The method ofclaim 9, wherein the sub-therapeutic dose of anti-VEGF to beadministered to the patient is determined by analyzing the level ofcirculating VEGF in the blood.
 15. The method of claim 9, wherein thesub-therapeutic dose of the anti-VEGF antibody is administered frombetween about 30 minutes to about 48 hours prior to administration ofthe albumin-bound chemotherapeutic/anti-VEGF antibody nanoparticlecomplexes.
 16. A method for enhancing the therapeutic outcome in apatient suffering from a cancer expressing soluble VEGF which patient isto be treated with nanoparticles comprising albumin-bound paclitaxel andanti-VEGF antibodies wherein said antibodies of the nanoparticles areintegrated onto and/or into said nanoparticles which method comprisestreating said patient with a sub-therapeutic amount of said anti-VEGFantibody.
 17. A method for enhancing the therapeutic outcome in apatient suffering from a cancer overexpressing soluble VEGF whichpatient has been treated with a sub-therapeutic amount of said anti-VEGFantibody said method comprising treating said patients with an effectiveamount of nanoparticles comprising albumin-bound paclitaxel andanti-VEGF antibodies wherein said antibodies of the nanoparticles areintegrated onto and/or into said nanoparticles.
 18. A unit-doseformulation of an anti-VEGF antibody which formulation comprises fromabout 1% to about 60% of a therapeutic dose of said antibody whereinsaid formulation is packaged so as to be administered as a unit dose.19. The formulation of claim 18, wherein the anti-VEGF antibody isbevacizumab or a biosimilar version thereof.
 20. The unit-doseformulation of claim 19, which formulation comprises from about 5% toabout 20% of a therapeutic dose of bevacizumab or a biosimilar versionthereof.
 21. A kit comprising: (a) an amount of an albumin-boundchemotherapeutic/anti-VEGF antibody complexes, (b) a unit dose of asub-therapeutic amount of anti-VEGF antibody, and optionally (c)instructions for use.
 22. The kit of claim 21, wherein the albumin-boundchemotherapeutic/anti-VEGF antibody complexes are lyophilized.